skip to main content
OSTI.GOV title logo U.S. Department of Energy
Office of Scientific and Technical Information

Title: Crystallographic snapshots of sulfur insertion by lipoyl synthase

Journal Article · · Proceedings of the National Academy of Sciences of the United States of America
 [1];  [2];  [3];  [2];  [2];  [3]
  1. Massachusetts Inst. of Technology (MIT), Cambridge, MA (United States); Pennsylvania State Univ., University Park, PA (United States); Univ. of Illinois at Urbana-Champaign, Urbana, IL (United States)
  2. Pennsylvania State Univ., University Park, PA (United States)
  3. Massachusetts Inst. of Technology (MIT), Cambridge, MA (United States)
+ Show Author Affiliations

Lipoyl synthase (LipA) catalyzes the insertion of two sulfur atoms at the unactivated C6 and C8 positions of a protein-bound octanoyl chain to produce the lipoyl cofactor. To activate its substrate for sulfur insertion, LipA uses a [4Fe-4S] cluster and S-adenosylmethionine (AdoMet) radical chemistry; the remainder of the reaction mechanism, especially the source of the sulfur, has been less clear. One controversial proposal involves the removal of sulfur from a second (auxiliary) [4Fe-4S] cluster on the enzyme, resulting in destruction of the cluster during each round of catalysis. Here, we present two high-resolution crystal structures of LipA from Mycobacterium tuberculosis: one in its resting state and one at an intermediate state during turnover. In the resting state, an auxiliary [4Fe-4S] cluster has an unusual serine ligation to one of the irons. After reaction with an octanoyllysine-containing 8-mer peptide substrate and 1 eq AdoMet, conditions that allow for the first sulfur insertion but not the second insertion, the serine ligand dissociates from the cluster, the iron ion is lost, and a sulfur atom that is still part of the cluster becomes covalently attached to C6 of the octanoyl substrate. Here, this intermediate structure provides a clear picture of iron–sulfur cluster destruction in action, supporting the role of the auxiliary cluster as the sulfur source in the LipA reaction and describing a radical strategy for sulfur incorporation into completely unactivated substrates.

Research Organization:
Argonne National Laboratory (ANL), Argonne, IL (United States)
Sponsoring Organization:
USDOE Office of Science (SC), Basic Energy Sciences (BES). Scientific User Facilities Division; National Institutes of Health (NIH); National Science Foundation (NSF); Meryl and Stewart Robertson Undergraduate Research Opportunities Program; Massachusetts Inst. of Technology Energy Initiative; DeFlorez Endowment; National Inst. of General Medical Sciences
Grant/Contract Number:
AC02-06CH11357; R01GM063847; R01GM103268; MCB-0543833; P41GM103403
OSTI ID:
1314240
Journal Information:
Proceedings of the National Academy of Sciences of the United States of America, Vol. 113, Issue 34; ISSN 0027-8424
Publisher:
National Academy of SciencesCopyright Statement
Country of Publication:
United States
Language:
ENGLISH
Citation Metrics:
Cited by: 62 works
Citation information provided by
Web of Science

References (34)

Biotin synthase mechanism: on the origin of sulphur journal November 1998
Characterization of RimO, a New Member of the Methylthiotransferase Subclass of the Radical SAM Superfamily journal October 2009
Crystal Structure of Biotin Synthase, an S-Adenosylmethionine-Dependent Radical Enzyme journal January 2004
Lipoyl Synthase Inserts Sulfur Atoms into an Octanoyl Substrate in a Stepwise Manner journal August 2006
Spectroscopic Changes during a Single Turnover of Biotin Synthase:  Destruction of a [2Fe-2S] Cluster Accompanies Sulfur Insertion † journal July 2001
A Serine → Cysteine Ligand Mutation in the High Potential Iron−Sulfur Protein from Chromatium vinosum Provides Insight into the Electronic Structure of the [4Fe−4S] Cluster journal January 1996
Features and development of Coot journal March 2010
Structure validation by Cα geometry: ϕ,ψ and Cβ deviation journal January 2003
PHENIX: a comprehensive Python-based system for macromolecular structure solution journal January 2010
PROCHECK: a program to check the stereochemical quality of protein structures journal April 1993
Crystal Structure and Stability Studies of C77S HiPIP:  A Serine Ligated [4Fe-4S] Cluster † journal January 2002
Evidence for a Catalytically and Kinetically Competent Enzyme–Substrate Cross-Linked Intermediate in Catalysis by Lipoyl Synthase journal July 2014
Expression, purification, and physical characterization of Escherichia coli lipoyl(octanoyl)transferase journal February 2005
MiaB, a Bifunctional Radical- S- Adenosylmethionine Enzyme Involved in the Thiolation and Methylation of tRNA, Contains Two Essential [4Fe-4S] Clusters † journal May 2007
Biotin:  A Timeless Challenge for Total Synthesis journal October 1997
Escherichia coli Lipoyl Synthase Binds Two Distinct [4Fe−4S] Clusters per Polypeptide † journal September 2004
Structural diversity in the AdoMet radical enzyme superfamily journal November 2012
Mechanistic Investigations of Lipoic Acid Biosynthesis in Escherichia coli :  Both Sulfur Atoms in Lipoic Acid are Contributed by the Same Lipoyl Synthase Polypeptide journal March 2005
Structures of lipoyl synthase reveal a compact active site for controlling sequential sulfur insertion reactions journal October 2014
Lipoyl Synthase Requires Two Equivalents of S -Adenosyl- l -methionine To Synthesize One Equivalent of Lipoic Acid † journal June 2004
Characterization of Lipoyl Synthase from Mycobacterium tuberculosis journal February 2016
Stable isotope studies on the biosynthesis of lipoic acid in Escherichia coli journal January 1980
Escherichia coli LipA Is a Lipoyl Synthase:  In Vitro Biosynthesis of Lipoylated Pyruvate Dehydrogenase Complex from Octanoyl-Acyl Carrier Protein † journal December 2000
Effect of Serinate Ligation at Each of the Iron Sites of the [Fe 4 S 4 ] Cluster of Pyrococcus furiosus Ferredoxin on the Redox, Spectroscopic, and Biological Properties † journal August 1999
Insight into the Polar Reactivity of the Onium Chalcogen Analogues of S-Adenosyl-l-methionine journal October 2004
S -Adenosylmethionine-Dependent Reduction of Lysine 2,3-Aminomutase and Observation of the Catalytically Functional Iron−Sulfur Centers by Electron Paramagnetic Resonance † journal February 1998
Solution NMR Study of the Electronic Structure and Magnetic Properties of Cluster Ligation Mutants of the Four-Iron Ferredoxin from the Hyperthermophilic Archaeon Pyrococcus furiosus journal October 1997
The Radical SAM Superfamily journal January 2008
Characterization of the Cofactor Composition of Escherichia coli Biotin Synthase † journal February 2004
Detailed assessment of X-ray induced structural perturbation in a crystalline state protein journal February 2010
Lipoyl Synthase Inserts Sulfur Atoms into an Octanoyl Substrate in a Stepwise Manner journal August 2006
PARP1 exhibits enhanced association and catalytic efficiency with γH2A.X-nucleosome journal December 2019
Cavin1 intrinsically disordered domains are essential for fuzzy electrostatic interactions and caveola formation journal February 2021
PHENIX: a comprehensive Python-based system for macromolecular structure solution. text January 2010

Cited By (16)

Enzymkaskadenreaktionen in der Biosynthese journal February 2019
Enzymatic Cascade Reactions in Biosynthesis journal May 2019
Anaerobic Radical Enzymes for Biotechnology journal April 2018
Lipoic acid journal September 2019
A unifying view of the broad-spectrum antiviral activity of RSAD2 (viperin) based on its radical-SAM chemistry journal January 2018
Following the electrons: peculiarities in the catalytic cycles of radical SAM enzymes journal January 2018
Structural and spectroscopic analyses of the sporulation killing factor biosynthetic enzyme SkfB, a bacterial AdoMet radical sactisynthase journal September 2018
The A-type domain in Escherichia coli NfuA is required for regenerating the auxiliary [4Fe–4S] cluster in Escherichia coli lipoyl synthase journal December 2018
On the Role of Additional [4Fe-4S] Clusters with a Free Coordination Site in Radical-SAM Enzymes journal March 2017
Lipoic Acid book January 1975
Lipoic Acid book June 2004
Insight into the reaction mechanism of lipoyl synthase: a QM/MM study journal December 2017
A Radical Clock Probe Uncouples H Atom Abstraction from Thioether Cross-Link Formation by the Radical S -Adenosyl- l -methionine Enzyme SkfB journal July 2018
Mechanistic Investigations of PoyD, a Radical S -Adenosyl- l -methionine Enzyme Catalyzing Iterative and Directional Epimerizations in Polytheonamide A Biosynthesis journal February 2018
Biochemical and Structural Characterization of a Schiff Base in the Radical-Mediated Biosynthesis of 4-Demethylwyosine by TYW1 journal May 2018
Mapping the key residues of SufB and SufD essential for biosynthesis of iron-sulfur clusters journal August 2017

Similar Records

Related Subjects

59 BASIC BIOLOGICAL SCIENCES
37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CHEMISTRY
iron–sulfur cluster
radical SAM enzyme
lipoic acid